A number of assays are currently available for the detection of target molecules in a sample. Some rely on the use of enzymes, and in particular enzymes that catalyse modifications to nucleic acids including the following discussed below.
Nucleases
Nucleases are enzymes that cleave phosphodiester bonds between the nucleotide subunits of nucleic acids. Deoxyribonucleases act on DNA while ribonucleases act on RNA, however some nucleases utilize both DNA and RNA as substrates. Nucleases can be further categorized as endonucleases and exonucleases, although some enzymes may have multiple functions and exhibit both endonuclease and exonuclease activity. Endonucleases cleave phosphodiester bonds within a polynucleotide chain. In contrast, exonucleases cleave phosphodiester bonds at the end of a polynucleotide chain. Exonucleases may remove ‘nucleotides from either the 5’ end or the 3′ end or from both ends of a DNA or RNA strand.
Non-limiting examples of protein exonucleases include Exonuclease I (E. coli), Exonuclease III (E. coli), Exonuclease VII and T7 Exonuclease. Exonuclease III (ExoIII) is an exonuclease that catalyzes the step-wise removal of mononucleotides from the 3′ end of blunt or 3′ recessed duplexed DNA. It is also capable of acting at nicks within duplexed DNA. ExoIII has minimal activity on single-stranded DNA or duplexed DNA that have single stranded protruding ends of at least 4 nt.
Catalytic Nucleic Acid Enzymes
Catalytic nucleic acid enzymes are non-protein enzymes capable of modifying specific substrates. Catalytic nucleic acid enzymes include DNA molecules (also known in the art as a DNAzyme, deoxyribozyme, or DNA enzyme), RNA, molecules (also known in the art as a ribozyme), and multi-component nucleic acid enzymes composed of multiple DNA and/or RNA molecules (also known in the art as an MNAzyme). Catalytic nucleic acid enzymes can modify specific nucleic acid substrate sequences by, for example, cleavage or ligation. A unique class of catalytic nucleic acid enzyme (known in the art as a horseradish peroxidase-mimicking DNAzyme) can catalyse peroxidase reactions that convert specific chemical substrates into their oxidated products which can for example, produce a change in colour or emit a fluorescent or chemiluminescent signal.
DNAzymes and ribozymes capable of cleaving or ligating RNA substrates, DNA substrates and/or chimeric DNA/RNA substrates, can generally only modify a target nucleic acid substrate that meets minimum sequence requirements. For example, the substrate should exhibit sufficient base pair complementarity to the substrate binding arms of the enzyme, and also needs a specific sequence at the site of catalytic modification. Examples of such sequence requirements at the catalytic cleavage site include the requirement for a purine:pyrmidine sequence for DNAzyme cleavage (10-23 model) and the requirement for the sequence uridine:X where X can equal A, C or U but not G, for the hammerhead ribozymes. The 10-23 DNAzyme is a DNAzyme that is capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds. This DNAzyme has a catalytic domain of 15 deoxyribonucleotides flanked by two substrate-recognition domains (arms).
MNAzymes are another category of catalytic nucleic acid enzymes. These multi-component nucleic acid enzymes require an assembly facilitator (e.g. a target nucleic acid) for their assembly and catalytic activity. MNAzymes are composed of multiple part-enzymes, or partzymes, which self-assemble in the presence of one or more assembly facilitators and form catalytically active MNAzymes capable of catalytically modifying substrates. The partzymes have multiple domains including sensor arms which bind to the assembly facilitator (such as a target nucleic acid); substrate arms which bind the substrate, and partial catalytic core sequences which, upon assembly of multiple partzyme components, combine to provide a complete catalytic core. MNAzymes can be designed to recognize a broad range of assembly facilitators including, for example, different target nucleic acid sequences. In the presence of the assembly facilitator, a catalytically active MNAzyme can assemble from partzyme components, and then bind and catalytically modify a substrate to generate an output signal. The assembly facilitator may be a target nucleic acid present in a biological or environmental sample. In such cases, MNAyme catalytic activity is indicative of the presence of the target. Several MNAzymes capable of cleaving nucleic acid substrates have been reported and additional MNAzymes which can ligate nucleic acid substrates are also known in the art.
Aptazymes are specific types of catalytic nucleic acids (DNAzymes, ribozymes or MNAzymes) which have been linked with an aptamer domain to allosterically regulate the nucleic acid enzymes such that their activity is dependent on the presence of the target analyte/ligand capable of binding to the aptamer domain. Complementary regulator oligonucleotides have been used to inhibit the activities of aptazymes in the absence of target analytes by binding to both the aptamer and part of the catalytic nucleic acid domains within the aptazymes. The inhibition of catalytic activity of aptazymes was reversible by the binding of target ligands to the aptamer portions thus promoting removal of the regulator oligonucleotide. The present inventors are not aware of any previous disclosure of a method that employs oligonucleotides designed to reversibly inhibit the modification of nucleic acid substrates by the catalytic activity of catalytic nucleic acids which are not coupled with an aptamer (i.e. a catalytic nucleic acid which is not an aptazyme), whereby inhibition is mediated by an oligonucleotide which binds to the catalytic core, or a portion thereof, of the catalytic nucleic acid.
Strand Displacing Polymerases
DNA polymerases catalyze the polymerization of deoxyribonucleotides into a DNA strand. They are naturally occurring enzymes responsible for DNA replication, in which the polymerase “reads” an intact DNA strand as a template and uses it to synthesize a new strand. Some DNA polymerases contain 5′-3′ proofreading exonuclease activity, whereby they will degrade any downstream strands encountered during synthesis (e.g. Taq DNA polymerase). In contrast, strand-displacing DNA polymerases have the ability to displace any downstream strands, encountered during synthesis. The downstream strands are not degraded and remain intact. Examples of strand-displacing DNA polymerases include Phi29, DNA polymerase I Klenow Fragment, VentR and Bst polymerase large fragment.
Kinases and Phosphatases
Kinases can catalyse the transfer of a γ-phosphate from ATP to specific amino acids in proteins and also to nucleic acid termini. Phosphatases can catalyse the removal of phosphate groups via hydrolysis of esterified phosphoric acid, resulting in a phosphate ion and a molecule with a free hydroxyl group. Protein phosphorylation via kinases and de-phosphorylation via phosphatases are important for regulating signal transduction pathways within a cell, thus performing crucial functions in cellular process such as metabolism, cell cycle progression, cell movement, and apoptosis. In molecular biology, phosphorylation of the 3′ termini of oligonucleotides can be used as a method to prevent their extension by polymerases. Alternatively, de-phosphorylation of the 3′ termini of oligonucleotides can be used as a method to allow for their extension by polymerases.
Target and Signal Amplification Technologies
In order to increase the sensitivity of target detection, strategies for target amplification or signal amplification have been employed, many of which utilize DNA and/or RNA polymerase enzymes. Examples of existing methods which employ target amplification include the polymerase chain reaction (PCR), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcript-mediated amplification (TMA); self-sustained sequence replication (3SR), or nucleic acid sequence based amplification (NASBA). Those methods which are dependent on strand displacement for amplification, for example SDA, RCA and LAMP, require the use of polymerases which possess strand displacing activity. Signal amplification cascades that utilize nucleases including nicking endonucleases have also been described (e.g. NESA).
Signal amplification cascades that utilize catalytic nucleic acids have also been described. One example involves the use of an oligonucleotide consisting of two adjacent peroxidase-mimicking DNAzymes joined by a ribonucleotide junction. The ends of this oligonucleotide are linked together via a short linker DNA which hybridizes to each end, so as to form a quasi-circular structure. The formation of this structure temporarily inhibits the catalytic activity of the DNAzymes. An MNAzyme that assembles in the presence of its target assembly facilitator molecule hybridizes directly to the DNAzymes and cleaves the ribonucleotide junction between them. Cleavage of the oligonucleotide containing the DNAzymes results in separation of the two DNAzymes from each other and from the short linker DNA resulting in activation of the DNAzymes. The limitations of this strategy are that amplification of signal is limited to only two DNAzymes activated for each MNAzyme cleavage event, and since these peroxidase-mimicking DNAzymes have no capacity to modify nucleic acid substrates, there is no mechanism for these DNAzymes to activate additional DNAzyme molecules. As such the strategy is unsuitable as a first step in a circular feedback cascade capable of exponential signal amplification. Further, the complementarity between the MNAzyme arms and the DNAzymes, which is necessary for the initial cleavage event to occur, may also result in the sequestering of DNAzyme molecules once they have been released from the quasi-circular structure, potentially limiting the sensitivity of the reaction.
Many limitations are evident in other existing signal amplification methods. For, example, in a number of techniques the speed of the reaction is limited by the number of target DNA molecules initially present in the sample. These and other methods thus often lack the speed and sensitivity required for clinical application. Others suffer from false positive signal generation in the absence of target due to inadequate inhibition of the catalytic nucleic acid molecules. A number of methods utilizing DNA strand displacement are slow and lack the sensitivity of target amplification methods. Assays relying predominantly on unique recognition sites can preclude detecting universal target sequences, or may require new probes for each new target sequence.
Thus, there is an ongoing need for methods for detecting and quantifying nucleic acid sequences and other targets which incorporate signal amplification.